One of the greatest challenges to maintaining the genetic integrity of an organism is the ability to recognize and repair DNA damage prior to the lesion being converted into a permanent mutation. The magnitude of this task and the exquisite level of discrimination that must be achieved to distinguish true catalytic substrates from nonsubstrate DNAs is underscored by the fact that these lesions occur infrequently amid vast excess of undamaged DNA. An additional complication is that the sites within DNA where recognition and chemistry must occur are buried within the DNA structure. To solve these respective challenges, DNA repair enzymes use both highly effective search strategies of bulk DNA and a process termed nucleotide flipping that moves bases out of the interior of the DNA into an active site pocket where repair chemistry occurs. Although nucleotide flipping is known to occur based on cocrystal structures, the fundamental basic processes that govern this mechanism are not known. This application proposes a comprehensive analyses of how nucleotide flipping occurs. The first specific aim will use a battery of complementary biophysical approaches to address a series of hypotheses that seek to determine how DNA glycosylases exploit the intrinsic properties of damaged DNAs that may ultimately lead to nucleotide flipping and release of the damaged base. Each hypothesis will be investigated by multiple techniques including fluorescence anisotropy, electron transfer within it-stacked DNA, fluorescence resonance energy transfer, differential electrophoretic mobilities of enzyme-DNA complexes and electron microscopy. Specific Aim 2 will include not only a full workup of the kinetics of nucleotide flipping using double and triple-labeled stopped flow analyses, but also computational molecular dynamics of the DNA helical parameters that enable these enzymes to distinguish substrate from nonsubstrate DNA. Overall, the effectiveness of this process is one of the major control points for the overall rate of mutagenesis within organisms.